In Vitro Implantation Model Using Human Endometrial SUSD2+

Objective This study evaluated a novel in vitro implantation model using human endometrial mesenchymal stem cells (EMSCs), SUSD2+, and myometrial smooth muscle cells (SMCs) that were co-cultured with mouse blastocysts as the surrogate embryo. Materials and Methods In this experimental study, SUSD2+ MSCs were isolated from human endometrial cell suspensions (ECS) at the fourth passage by magnetic-activated cell sorting. The ECS and SUSD2+ cells were separately co-cultured with human myometrial muscle cells for five days. After collection of mouse blastocysts, the embryos were placed on top of the co-cultured cells for 48 hours. The interaction between the embryo and the cultured cells was assessed morphologically at the histological and ultrastructural levels, and by expression profiles of genes related to implantation. Results Photomicrographs showed that trophoblastic cells grew around the embryonic cells and attached to theECS and SUSD2+ cells. Ultrastructural observations revealed pinopode and microvilli-like structures on the surfaces of both the ECS and SUSD2+ cells. Morphologically, the embryos developed to the egg-cylinder stage in both groups. Gene expression analysis showed no significant differences between the two groups in the presence of an embryo, but an increased expression of αV was detected in SUSD2+ cells compared to ECS cells in the absence of an embryo. Conclusion This study showed that SUSD2+ cells co-cultured with SMCs could interact with mouse embryos. The co-cultured cells could potentially be used as an implantation model.


Introduction
Implantation results from successful interactions between the embryo and endometrial epithelium during the mid-secretory phase of the menstrual cycle when the endometrium is receptive. At this so-called "window of implantation", ultrastructural alterations occur on the surface of endometrial epithelial cells and serve as important implantation markers of the receptive endometrium (1,2).
Human implantation proceeds through three main stages: apposition, adhesion, and invasion. During the apposition stage, the blastocyst interacts with the apical surface of the luminal epithelium through two-way molecular communication. During the receptive phase, the luminal epithelial surface changes from a non-adhesive to adhesive surface, which results in the appearance of pinopodes and reduction of lateral junctional complexes. During attachment, the embryo initiates a physical connection with the apical surface of the endometrial epithelium; however, during invasion, the trophoblast cells penetrate between the epithelial cells, migrates to and invades the blood vessels (3). Impairment of implantation is considered a major cause of human pregnancy loss and infertility in assisted reproductive technologies (ART) (4,5). Improving ART outcomes and preventing early pregnancy loss requires a better understanding of the mechanisms of interactions between the embryo and the endometrium during the implantation process. Since the in vivo study of human embryo implantation is unethical and has limitations, and the results of studies performed in animal models are not always applicable in humans, in vitro implantation models using human cells provide an alternative approach (6).
In vitro implantation models are categorized into several types (6). One mainly focuses on the interaction between endometrial epithelial cells and the embryo to evaluate the early stages of implantation (6)(7)(8). In another group of implantation models, late stages of implantation are studied through two-dimensional culture of endometrial stromal cells with an embryo (6,9). In more complex models, endometrial epithelial and stromal cells are cocultured with an embryo in a three-dimensional culture system, allowing the study of both early and late stages of implantation (6,(10)(11)(12). Because of limited access to human embryos, a number of studies have used surrogate embryos in designing implantation models (6). Several have employed mouse blastocysts (13,14), while most have used trophoblast spheroids derived from trophoblastic cell lines (15,16).
The human endometrium is a dynamic tissue that undergoes cyclical shedding and regeneration during each reproductive cycle. The identification of rare populations of adult stem cells in both the stratum functionalis and basalis suggest that they may play a critical role in endometrial regenerative activities (17)(18)(19). Endometrial stem/progenitor cells have adult stem cell characteristics of clonogenicity, high proliferative potential and multilineage differentiation potential (17,20). They comprise epithelial, mesenchymal, and endothelial stem/ progenitor cells. Endometrial mesenchymal stem cells (EMSCs) are located ina perivascular region, and include pericytes and perivascular cells (21). They are identified by specific markers, such as co-expression of CD146 and PDGF-Rβ and a single marker, SUSD2 (W5C5) (17,18,(22)(23)(24)(25)(26).
EMSCs have the potential to differentiate into several cell types in vitro (18,26); thus, they may have extensive applications in cell therapy, tissue reconstruction, and regenerative medicine (27,28). There are limited reports regarding the differentiation of endometrial stem/progenitor cells into endometrial glands and epitheliaupon transplantation under the kidney capsules of immunodeficient mice (29). Recently, we showed that CD146 + cells isolated from human endometrium differentiated into endometrial epithelial-like cells during co-culture with myometrial smooth muscle cells (SMCs) (30). Campo et al. (31) demonstrated that transplantation of cultured human endometrial side population (SP) cells, which were comprised of stromal and epithelial cells, to a decellularised porcine uterus resulted in some recellularisation with human vimentin positive stromal cells and rare cytokeratin positive epithelial cells. Recently, López-Pérez et al. (28) reported that injection of a human endometrial SP under kidney capsules induced reformation of human endometrium, which was confirmed by the presence of typical endometrial markers. They concluded that these cells had the optimum capacity to regenerate endometrial-like tissue.
Despite the differentiation potential of adult stem cells to endometrial-like cells, and according to our knowledge, few studies have designed an in vitro implantation model by using these cell types. Thus, the aim of the present study was to evaluate a novel in vitro implantation model that mimics the in vivo condition by using human EMSCs co-cultured with human myometrial SMCs to assess implantation with mouse blastocysts as the surrogate embryo.

Materials and Methods
All reagents were purchased from Sigma Aldrich (Germany) unless otherwise indicated.

Human tissue collection
For this experimental study, human endometrial (n=10) and myometrial (n=10) tissues were obtained from healthy fertile women (aged 25-40 years) during the proliferative phase, and who were undergoing hysterectomies for non-pathological conditions. The women had not taken any exogenous hormones for three months before surgery (Table S1, See Supplementary Online Information at www.celljournal.org). Samples were transported to the laboratory in equilibrated and prewarmed Leibovitz'sL-15 medium supplemented with 10 mg/ml human serum albumin, 100 IU/ml penicillin and 100 μg/ml streptomycin within 1-2 hours.
The Ethics Committee of the Medical Faculty of Tarbiat Modares University (Tehran, Iran, no.1394.137) approved this experimental study and written informed consent was received from all patients. Figure S1 (See Supplementary Online Information at www.celljournal.org) shows the experimental design. Human endometrial cells were isolated mechanically and enzymatically from endometrial tissues and cultured up to the fourth passage. Then, the SUSD2 + cells were sorted by magnetic activated cell sorting (MACS) and their characteristics were confirmed by immunohistochemistry. The endometrial cell suspensions (ECS) and sorted SUSD2 + cells were separately co-cultured with myometrial smooth muscle for five days, after which the cultivation period was extended for an extra 48 hours in the presence or absence of mouse blastocysts in order to establish two in vitro embryo implantation models. At the end of the culture periods, the endometrial (ECS and SUSD2 + cells) and embryonic cell interactions were assessed by morphological, ultrastructural and molecular studies.

Morphological evaluations of endometrial and myometrial samples
Ten samples each of endometrial and myometrial tissue were separately fixed in Bouin's solution, processed, embedded in paraffin wax and sectioned into 7 µm thicknesses. After hematoxyline and eosin (H&E) staining, the sections were observed with a light microscope and their normal morphology was evaluated (32).

Isolation of human endometrial cells
Human endometrial cells were isolated from tissues as per the Chan et al. (33) method. Briefly, human endometrial tissue was washed in phosphate-buffered saline (PBS) and then cut into small 1×1 mm pieces within Dulbecco's modified Eagle's Medium/Hams F-12 (DMEM/F-12) that contained 100 mg/ml penicillin G sodium and 100 mg/ml streptomycin sulphate B. The tissue fragments were separated into single cells using collagenase type 1 (300 μg/ml) and deoxyribonuclease type I (40 μg/ ml) for 90 minutes together with a mechanical method. To eliminate glandular and epithelial components, the cell suspension was passed sequentially through sieves of mesh at sizes of 100 and 40 µm (SPL Life Sciences Implantation Model Using SUSD2 + Cells and Mouse Embryo Co., Korea), respectively (34). Endometrial stromal cells in the supernatant were cultured using DMEM/F-12 that contained antibiotics and 10% fetal bovine serum (FBS, all from Invitrogen, UK) and incubated at 37˚C in 5% CO 2 . The cells were cultured up to passaged when they reached to 80-100% confluency, used for the following assessments.

Confirmation of endometrial mesenchymal cells using flow cytometry
A number of the passage-4 endometrial cells were evaluated for mesenchymal (CD90, CD73 and CD44) and hematopoietic markers (CD45 and CD34) by flow cytometric analysis. A total of 1×10 5 endometrial cells were suspended in 50 μl of PBS and incubated with direct fluorescein isothiocyanate (FITC)-conjugated antibodies (anti-human CD90, CD44, and CD45, 1:50 dilutions) and direct phycoerythrin (PE)-conjugated antibodies (antihuman CD73 and CD34; 1:50 dilutions) at 4˚C for 45 minutes. Finally, 200 μl of PBS was added and the cells were examined with a FACSCaliburcytometer (Becton Dickinson, Germany). The flow cytometric analysis was repeated three times.

SUSD2 + cell isolation by magnetic-activated cell sorting
After the fourth passage, the cultured human endometrial cells were washed, resuspended (up to 1×10 7 cells/100 μl) in cold PBS and incubated with mouse anti-SUSD2 monoclonal antibody (327401, 8:200, Biolegend, UK) at 4˚C for 30 minutes. The cells were washed with MACS separation buffer (130-091-221, Miltenyi Biotec, Germany), then they were incubated with goat antimouse IgG Microbeads antibody (130047102, 20:100, Miltenyi Biotec, Germany) at 4˚C for 20 minutes. The cell suspensions were washed and run through the MACS column, followed by washing the column for three times with 500 μl MACS separation buffer. Magnetically labelled cells (SUSD2 + ) were mostly retained on the column and the unlabelled cells (SUSD2 -) were eluted. Trypan blue staining (0.4%) was performed to determine SUSD2 + cell viability following MACS sorting. All experiments were repeated three times.

Immunocytochemistry of sorted endometrial SUSD2 + cells
The purity of the magnetic bead-sorted human endometrial (SUSD2 + ) cells was assessed by immunocytochemistry (n=3 samples). These cells were incubated with mouse anti-SUSD2 monoclonal antibody (327401, 8:200, Biolegend, UK) at 4˚C for 30 minutes. After washing the cells with PBS, they were incubated with secondary goat anti-mouse polyclonal antibody conjugated with Alexa Fluor® 488 (405319, 1:100 in PBS, Biolegend, UK) for 2 hours at 37˚C and washed three times with PBS. Nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI, D9542, Sigma, Germany) for 30 seconds. For negative controls, the cells were treated with the 10% unimmunized mouse serum in PBS instead of primary antibody. All experiments were repeated three times.

In vitro culture of human myometrial cells
After dissection, the tissue fragments of the myometrium were cultured according to the explant method as reported by Fayazi et al. (30). Briefly, the human myometrial tissues (n=10) were washed with PBS and then cut into 1×1 mm pieces in DMEM/F-12 that contained 100 mg/ml penicillin G sodium and 100 mg/ml streptomycin sulphate B. Finally, the fragments were placed in each well and the emerging cells were allowed to grow in complete DMEM/F-12 supplemented with 10% FBS to confluency at 37˚C and 5% CO 2 for three weeks. The medium was changed every two days. The characteristics of isolated myometrial cells were confirmed by immunocytochemical analysis.

Immunocytochemistry of myometrial cultured cells
Passage-2 trypsiniszed myometrial cells (n=3 samples) were cultured on cover slips. After attachment, the cultured cells were washed three times with PBS, fixed with 4% paraformaldehyde at 4˚C for 20 minutes, and permeabilised with 0.3% TritonX-100 for 45 minutes. Non-specific binding was blocked with 10% normal goat serum in PBS. Cells were separately incubated with the SMC markers, mouse anti-vimentin monoclonal antibody (V6389, 3:100 in PBS, Sigma-Aldrich, Germany) and rabbit anti-alpha smooth muscle actin polyclonal antibody (ab5694, 1:100 in PBS, Abcam, UK) at 4˚C overnight. The cells were washed in PBS three times, and incubated with secondary antibodies rabbit anti-mouse polyclonal antibody conjugated with Texas red (315-075-003, 3:100 in PBS, Biolegend, UK) and goat anti-rabbit IgG conjugated with FITC (ab6717, 1:1000 in PBS, Abcam, UK) at 37˚C for 2 hours. For negative controls, 10% unimmunized mouse serum in PBS was used instead of primary antibody. The immunocytochemistry analysis was repeated three times.

Implantation models using SUSD2 + cells and endometrial cell suspensions
The SUSD2 + cells (group 1) and ECS (group 2) were separately co-cultured with myometrial cells as two experimental groups. In each group, 10 4 SUSD2 + or ECS cells were cultured in 48-well plates with 5×10 3 myometrial cells per well for five days. On the fifth day of culture, the mouse blastocysts were placed on the top of each well, with n=5 embryos in each well and a total of 45 embryos in each group for at least 9 repeats. The groups co-cultured in the absence of mouse blastocysts were considered to be the control groups. Then, these cells were cultured and monitored up to an additional 48 hours and evaluated morphologically by inverted microscope, live/dead staining, scanning electron microscope (SEM) and analysis of gene expressions related to implantation.

Live/dead staining
We assessed the viability of the embryos and cells at 48 hours after the embryo culture on the top of each of the co-culture experimental groups by using a live/dead viabilitykit (L-3224, Invitrogen, UK). For this purpose, the cells were incubated with calcein AM (green) and ethidium homodimer-1 (EthD-1, red) for intracellular esteraseactivity and plasma membrane integrity, respectively, according to the manufacturer's instructions. Then, the embryos and cells were observed under a fluorescent microscope (Nikon TE2000, Japan). This experiment was performed in triplicate.

Scanning electron microscope
After two days of co-culture of the experimental groups with embryos, we examined the ultrastructure and interaction of the implanted embryos with co-cultured cells by SEM and compared them with their respective controls (groups without embryos). The specimens were fixed in 2.5% glutaraldehyde and post-fixed with 1% osmium tetroxide in PBS for two hours. After dehydration in an ascending ethanol series, the specimens were dried in a freeze dryer (Snijders Scientific LY5FME, Netherlands), mounted and coated with gold particles (BalTec, Switzerland) and examined under SEM (Philips XL30, Netherland). These experiments were repeated three times.

Expression of implantation genes by real-time reverse transcription polymerase chain reaction
We evaluated the expressions of genes related to implantation: αV and β3 integrin, interleukin-1 receptor (IL-1R), leukaemia inhibitory factor (LIF) and LIF receptor (LIFR). Total RNA was extracted from the collected cells after seven days of co-culture in both groups in the presence and absence of mouse embryos (5 embryosper well and, in total, 15 embryos per group with at least 3 replicates) using TRIzol (Invitrogen, UK). The concentration of isolated RNA was determined by a spectrophotometer, then cDNA was synthesized using a cDNA kit (Thermo Scientific, Lithuania, EU) in a total volume of 20 μl and the samples were stored at -80˚C until analysis. As shown in Table 1, the primers were designed based on human mRNA coding sequences using GenBank (http://www.ncbi.nlm.nih.gov) and synthesized at CinnaGen Company (Iran). The β-actin gene was used as an internal control.

Light microscopic observation of implantation models
Phase contrast imaging of implantation models using mouse blastocyst in studied groups were demonstrated in Figure 3A-F. The morphology of ECS and SUSD2 + cells co-cultured with myometrial SMCs without embryos showed a flattened monolayer of spindleshaped cells after the cultivation period (Fig.3, first  column).
However, the implanted mouse embryos incubated with the co-cultures demonstrated similar morphological features between the ECS and SUSD2 + groups. The trophoblastic cells migrated from the embryos and proliferated, and the embryonic cells spread on the endometrial/myometrial cell layer and were tightly attached (Fig.3, second column).
The vital live/dead staining of the embryos on the co-cultured cells shows that all of the mouse implanted embryos were viable after 48 hours of culture (Fig.3, third column).

Electron microscopic observation of implantation models
SEM evaluation of mouse blastocyst implantation on top of the ECS co-cultured with myometrial SMCs and the SUSD2 + cells co-cultured with myometrial SMCs are shown in Figure 4A-E and F-J, respectively. Ultrastructural evaluation of the human ECS or SUSD2 + cells co-cultured with human myometrial cells demonstrated that both had similar flattened spindle-shaped and flattened cells attached to the plate (Fig.4A, F). Some surface apical projections were seen on the endometrial cells adjacent to the implanted embryos, and these projections were similar to pinopodes (red arrowhead, Fig.4D, I) and microvilli (yellow arrowhead, Fig.4D, I).
The images obtained from the SEM indicated vertical growth of the embryos and the formation of mouse egg-cylinders in both studied groups. However, two different morphologies related to implanted embryos were observed at the ultrastructural level in each group: one with the presence of polarized cells (epiblast cells) arranged radially around the lumen of the proamniotic cavity and the other without polarized cells. This observation showed embryonic development on these co-cultures. Figure 4K shows a comparison of the ratios of gene expressions related to implantation (αV, β3, IL-1R, LIF and LIFR) to β-actin in both implantation models to the expression of β-actin in both implantation models in the absence or presence of embryos.
The expression of genes related to implantation was not significantly different between the groups in the presence and absence of mouse embryos.

Discussion
Considering the differentiation potential of EMSCs, SUSD2 + stem cells were used in the present study, for the first time, to create a new model of embryo implantation in comparison with an endometrial cell suspension that used mouse blastocysts as the surrogate embryo. For this purpose, SUSD2 + mesenchymal stem cells were isolated and co-cultured with SMCs and mouse blastocysts. Our results at the morphological and ultrastructural levels showed that the mouse blastocysts could interact with ECS and SUSD2 + cells and advance through the early stages of in vitro development within 48 hours. Moreover, electron micrographs indicated the ultrastructural changes in endometrial epithelial-like cells, including the appearance of pinopode-like and microvilli-like structures that are markers for early stages of implantation.
In another point of view, the ultrastructure of mouse embryos in the present study indicated the progression of their developmental stages and the formation of an eggcylinder. This stage of in vitro development is observed before gastrulation in mouse embryos (37,38).
Evaluation of the expression of genes related to implantation in ECS and SUSD2 + cells after co-culture with SMCs indicated that these genes were expressed. Moreover, there was an increase in the expression of αV in SUSD2 + cells compared to ECS. No significant differences were observed in the expressions of the other genes (β3, IL-1R, LIF and LIFR) between these groups. These data showed that SUSD2 + EMSCs are multipotential cells that could differentiate to endometriallike cells. Similarly, Fayazi et al. (30) revealed that CD146 + endometrial cells could express genes related to implantation, including secreted phosphoprotein 1 and matrix metalloproteinase-2, after differentiation into epithelial-like cells. In agreement, Lü et al. (11) showed that, after co-culturing endometrial epithelial and stromal cells with SMCs, the reconstructed tissue expressed β3 integrin, heparin-binding epidermal growth factor-like growth factor, and HOXA-10.
Our results showed no significant differences between Implantation Model Using SUSD2 + Cells and Mouse Embryo the studied groups in the presence and absence of mouse embryos regarding the expression of genes related to implantation. It seems that epithelial-like cells derived from SUSD2 + stem cells and ECS in the presence of mouse embryo exhibit the same gene expression profile as that in the absence of an embryo. Thus so far, no evidence has been reported to evaluate the effects of embryos on the expression of genes related to implantation in cultured endometrial stem cells. In relation to this, Popovici et al. (39) have reported that co-culture of trophoblast with endometrial stromal cells reduces the expression of matrix metalloproteinase-11 and increases the expression of IL-1 receptors in these cells. It has been suggested that the difference in the species sources of embryo and cultured cells (human endometrial cells and mouse embryos) in our study can affect the expression pattern profile of genes related to implantation and/or the expression of these genes may be time-dependent. Considering that implantation has a wide genomic profile, gene expression analyses in this study were not timed according to their in vivo time of expression. Moreover, possibly during the expansion of SUSD2 + cells in culture, they undergo some changes depending on cell density, cell-cell contact, and Notch signalling (40). In the present study, the endometrial tissue samples were collected from a population between 25 and 40 years of age. It should be mentioned that the age of human samples as a source of the endometrial cells might affect embryo implantation and the expressions of genes related to implantation. Nevertheless, due to a limited sample size and some limitations to prepare more human tissue in this study, this should be considered in further investigations.

Conclusion
This study showed that SUSD2 + cells during co-culture with SMCs can interact with mouse embryos. These co-cultured cells have the potential to be used as an implantation model.